Color Your Classroom: Engaging Students with Bacteria and Bio-Art
Transformation Troubleshooting TRANSFORMATION TROUBLESHOOTING GUIDE
PROBLEM:
ANSWER:
CAUSE:
Continue to incubate source plate at 37ºC for a total of 16-20 hours.
Incubation time too short
Poor cell growth on source plate
When pouring plates, be sure to add antibiotics & additives at the correct step.
Antibiotic added to source plate
Use a thermometer to check incubator temperature. Adjust temp. to 37°C if necessary. Ensure the correct concentration of antibiotic was added to plates - Make sure ReadyPour is cooled to 60°C before adding antibiotic.
Incorrect incubation temperature
Incorrect concentration of antibiotics in plates
Satellite colonies seen on transformation plate
Make sure ReadyPour is cooled to 60°C before adding antibiotic.
Antibiotic is degraded
Plates were incubated too long
Incubate the plates overnight at 37ºC (24 hours).
Allow cell suspension to fully absorbed into the medium before inverting plates.
Plates containing transformants were inverted too soon
Colonies appeared smeary on transformation plate
After pouring plates, allow them dry overnight at room temp. Alternatively, warm plates at 37°C for 30 min. before plating cells
Experimental plates too moist
Ensure plasmid DNA was added to transformation tube.
Plasmid DNA not added to transformation mix
Make sure that pipets are used properly. If using micropipets, make sure students practice using pipets
Incorrect host cells used for transformation
Confirm that correct bacterial strain was used for transformation
No colonies seen on transformation plates
Ensure that temp. was 42ºC & heat shock step took place for no more than 90 seconds.
Cells were not properly heat shocked
Incorrect antibiotics
Be certain that the correct antibiotic was used.
Completely resuspend the cells in the CaCl 2 , leaving no cell clumps (vortex or mix vigorously to fully resuspend cells). Cell suspension should be cloudy.
Cells not well resuspended in CaCl 2
Not enough cells used for transformation
Pick more colonies from source plate (1-2 colonies @ 1-2 mm width per 500µl CaCl 2 )
Low transformation efficiency
Important that source cells grow no longer than 20 hrs. Refrigerate plates after 20 hrs if necessary. Do not use source plates that have been incubated longer than 24 hours, refrigerated or not).
Source plates were incubated for more than 20 hours
Prepare transformation plate and use shortly after preparation
Experimental plates too old
Completely resuspend the cells in the CaCl 2 , leaving no cell clumps (vortex or mix vigorously to fully resuspend cells). Cell suspension should be cloudy.
Cells not well resuspended in CaCl 2
CaCl 2 solution not cold enough
Pre-chill CaCl 2 before adding cells to the CaCl 2
Extend incubation of celll suspension on ice 10-15 min. (should not exceed 30 min. total). This increases the transformation efficiency.
Cell solution not cold enough
Ensure that correct volume of plasmid was added to the transformation tube. If using micropipets, make sure students practice using pipets.
Too much or too little plasmid DNA added to cell suspension
Ensure that temperature was 42ºC and that heat shock step took place for no more than 90 seconds.
Cells were not properly heat shocked
Make sure ReadyPour is cooled to 60°C before adding antibiotic.
Antibiotics were degraded prior to pouring plates
Ensure that the correct concentration of antibiotic was used
Incorrect concentration of antibiotics in plates
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